Figure 8

MdtM-catalysed Rb ⁺ /H ⁺ , Li ⁺ /H ⁺ and Ca ²⁺ /H ⁺ exchange at alkaline pH. Exchange was determined by the fluorescence dequenching of acridine orange in inverted vesicles derived from antiporter-deficient E. coli TO114 cells that overexpressed recombinant wild-type MdtM (black traces) or the dysfunctional MdtM D22A mutant (grey traces). A ΔpH across the vesicle membrane was established by addition of lactate as indicated and once the fluorescence quench of acridine orange achieved a steady state, 40 mM Rb₂SO₄ (A), 40 mM Li₂SO₄ (B) or 40 mM CaSO₄ (C) was added to the vesicles. Addition of 100 μM CCCP abolished the ΔpH. The fluorescence intensity of each measurement is represented as a percentage of the initial acridine orange fluorescence signal prior to addition of lactate. The fluorescence measurements were conducted at pH 9.0 and the traces shown are representative of experiments performed in triplicate on at least two separate preparations of inverted vesicles.